Compositions and methods of cell attachment

ABSTRACT

Compositions, devices and methods are described for improving adhesion, attachment, and/or differentiation of cells in a microfluidic device or chip. In one embodiment, one or more ECM proteins are covalently coupled to the surface of a microchannel of a microfluidic device. The microfluidic devices can be stored or used immediately for culture and/or support of living cells such as mammalian cells, and/or for simulating a function of a tissue, e.g., a liver tissue, muscle tissue, etc. Extended adhesion and viability with sustained function over time is observed.

FIELD OF THE INVENTION

The present invention contemplates compositions, devices and methods ofimproving adhesion, attachment, and/or differentiation of cells in amicrofluidic device or chip. In one embodiment, ECM protein(s) iscovalently coupled to the surface of a microchannel of a microfluidicdevice. The microfluidic devices can be either stored, or immediatelyused for culture and/or support of living cells such as mammalian cells,and/or for simulating a function of a tissue, e.g., a liver tissue,muscle tissue, etc. Extended adhesion and viability with sustainedfunction over time is observed.

BACKGROUND

Cell adhesion is a central mechanism that ensures the structuralintegrity of tissue and is often a requirement for its biologicalfunction. The most prominent cell-matrix adhesion structures areso-called focal contacts. Focal contacts consist of large patches oftransmembrane adhesion receptors from the integrin-family. Theseintegrin patches in the cell membrane can reach lateral sizes of severalmicrometers. On the extracellular side, integrin binds to ligands suchas the ECM proteins collagen, fibronectin and vitronectin. On theintracellular side, the receptors are linked to the actin cytoskeletonvia a cytoplasmic plaque composed of many different proteins, includingtalin, vinculin, paxillin, and α-actinin. See Zamir and Geiger,“Molecular complexity and dynamics of cell-matrix adhesions,” J. CellScience 114: 3583 (2001). This connection to the cytoskeleton, which isoften organized in the form of stress fibers, allows transmitting forcesbetween cells and the ECM through focal contacts. Adhesions betweencells and the extracellular matrix (ECM) are known to modulate numerouscellular events.

Cell adhesion is also important in cell culture. Researchers haveattempted to optimize the culture conditions of cells by extracellularmatrix (ECM) coating of the culture dish, culture well, or culturechannel. However, these ECMs are often applied generically with mixedresults, depending on the cell type and culture conditions. What isneeded is a more specific use of ECMs in the context of specific celltypes and culture conditions.

SUMMARY OF THE INVENTION

The present invention contemplates compositions, devices and methods ofimproving adhesion, attachment, differentiation, longevity, quiescence,or biological function of cells in a microfluidic device or chip. In oneembodiment, one or more proteins (e.g. ECM proteins) or peptides (e.g.RGD) are covalently coupled to the surface of a microfluidic device,whether within a microchannel or an open structure. The microfluidicdevices can stored and used later, or they can be immediately used forculture and/or support of living cells such as mammalian cells, and/orfor simulating a function of a tissue, e.g., a liver tissue, muscletissue, etc., or simulating a function of an organ, e.g., a Liver-Chip,a Lung-Chip, etc. Extended adhesion and viability with sustainedfunction over time is observed. In one embodiment, the microchannelcomprises a surface comprising a silicone polymer. In one embodiment,the silicone polymer is polydimethylsiloxane or “PDMS,” In oneembodiment, the ECM protein is covalently coupled to a PDMS surfaceusing a crosslinker, such as the heterobifunctional linkerN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate(Sulfa-SANPAH). In one embodiment, the living cells are exposed to fluidflow, the fluid flow providing shear stress.

In one embodiment, the present invention contemplates a method ofculturing cells, comprising: a) providing a microfluidic devicecomprising a microchannel comprising a surface, said microchannel influidic communication with a fluid source comprising fluid; b)covalently attaching one or more proteins or peptides to saidmicrochannel surface so as to create a treated surface; c) seedingviable cells on said treated surface so as to create attached cells; c)flowing fluid from said fluid source through said microchannel so as tocreate flowing conditions; and d) culturing said attached cells undersaid flow conditions such that said cells remain attached and viable(e.g. viable for at least 14 days). It is not intended that the presentinvention be limited to any particular cell type; a variety of celltypes are contemplates (including more than one cell type). In oneembodiment, said cells are hepatocytes. It is not intended that thepresent invention be limited to any particular protein or peptide; avariety are contemplated, including mixtures. For example, in oneembodiment, the covalently attached protein is collagen. In anotherembodiment, a mixture of proteins are covalently attached, e.g. amixture of collagen type I, fibronectin and collagen type IV. In yetanother embodiment, the RGD peptide is attached (or a peptide comprisingthe RGD motif is attached). In one embodiment, the microchannel furthercomprises a membrane. In one embodiment, the membrane comprises PDMS. Inone embodiment, the membrane comprises a crosslinker (e.g. covalentlybound to the membrane). In one embodiment, the crosslinker is abifunctional crosslinker. In one embodiment, the crosslinker isSulfo-SANPAH (which is light activated with UV irradiation). In oneembodiment, an extracellular matrix protein (e.g. laminin) is attachedto the crosslinker (e.g. covalently bound) so as to provide anECM-coated membrane. In one embodiment, the irradiated membrane iswashed before the ECM attachment step. In one embodiment, the viablecells are further seeded onto the ECM-coated membrane. In oneembodiment, the viable cells are neurons. In one embodiment, the viablecells are motor neurons. In one embodiment, the viable cells arehepatocyte. In one embodiment, the viable cells are muscle cells. In oneembodiment, the viable cells are skeletal muscle cells. In oneembodiment, the skeletal muscle cells are human.

In one embodiment, the microchannel further comprises a micropatternedmembrane. In one embodiment, the micropatterned membrane comprises PDMS.In one embodiment, a bifunctional crosslinker is attached to themicropatterned membrane. In one embodiment, the micropatterned membraneis in the flow channel of a microfluidic device. In one embodiment, themicropattern is parallel to the fluid flow. In one embodiment, themicropattern is perpendicular to the fluid flow. In one embodiment, anextracellular matrix protein (e.g. laminin) is attached to thecrosslinker (e.g. covalently bound) so as to provide an ECM-coatedmicropatterned membrane. In one embodiment, the viable cells are furtherseeded onto the ECM-coated micropatterned membrane. In one embodiment,the viable cells are neurons. In one embodiment, the viable cells aremotor neurons. In one embodiment, the viable cells are hepatocyte. Inone embodiment, the viable cells are muscle cells. In one embodiment,the viable cells are skeletal muscle cell. In one embodiment, theskeletal muscle cells are human (“hSKMCs”). In one embodiment, theskeletal muscle cells elongate in the grooves of the micropatternedmembrane.

In one embodiment, the crosslinker is only attached to a portion of themembrane or the micropatterned membrane. In one embodiment, the portionwhere the crosslinker is not attached is covered with a mask (e.g. thecrosslinker is light activated and the mask blocks the light) and theportion where the crosslinker is attached is unmasked. The mask may beadhesive material (e.g. tape) or non-adhesive material (e.g. metal ormetal foil such as aluminum foil). In one embodiment, an extracellularmatrix protein (e.g. laminin) is attached to the crosslinker (e.g.covalently bound) in the unmasked portion so as to provide an ECM-coatedmembrane or micropatterned membrane. In one embodiment, the viable cellsare further seeded onto the ECM-coated portion of the membrane ormicropatterned membrane. In one embodiment, the viable cells areneurons. In one embodiment, the viable cells are motor neurons. In oneembodiment, the viable cells are hepatocyte. In one embodiment, theviable cells are muscle cells. In one embodiment, the viable cells areskeletal muscle cell. In one embodiment, the skeletal muscle cells arehuman.

It is not intended that the present invention be limited to the methodby which micropatterns are introduced into the membrane. In oneembodiment, the micropatterns are introduced into the membrane throughthe use of an existing micropatterned silicon wafer mold; PDMS materialcan be spin coated on the mold and cured (the membrane is thereafterremoved from the mold and used to assemble a microfluidic device orchip). In one embodiment, the micropattern is embossed using an existingmicropatterned silicon wafer using both heat and pressure.

The present invention contemplates that in certain embodiments, thesurface can be treated prior to step b). A variety of surface treatments(e.g. chemical vapor deposition, plasma oxidation, Corona, RF plasma,etc.) are possible. For example, in one embodiment, the presentinvention contemplates wherein said microchannel surface is PDMS andwherein said PDMS is plasma treated prior to step b).

It is not intended that the present invention be limited by the mannerin which the proteins or peptides are covalently attached. In oneembodiment, a crosslinker is used. In another embodiment, a bifunctionalcrosslinker is used. In a preferred embodiment, the protein or peptideis covalently attached to said microchannel surface usingN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

The present invention contemplates that the cells will be remain viableand can be tested to confirm this. However, it is not intended that thepresent invention be limited to a particular viability test. In oneembodiment, the method further comprises e) assessing viability bymeasuring the level of activity of one or more cellular enzymes. Avariety of enzymes can be used for this purpose, including but notlimited to, a CYP enzyme, a transaminase and the like. In anotherembodiment, the present invention contemplates that the method furthercomprises e) assessing viability by measuring the level of expression ofone or more cellular proteins.

In yet another embodiment, the present invention contemplates a methodof culturing specific cells, comprising: a) providing a microfluidicdevice comprising a microchannel comprising a surface, said microchannelin fluidic communication with a fluid source comprising fluid; b)covalently attaching one or more proteins or peptides to saidmicrochannel surface so as to create a treated surface; c) seedingviable hepatocytes on said treated surface so as to create attachedcells; e) flowing fluid from said fluid source through said microchannelso as to create flowing conditions; and d) culturing said attached cellsunder said flow conditions such that said cells remain attached andviable (e.g. viable for at least 14 days). It is not intended that thepresent invention be limited by the nature or species of hepatocytes. Inone embodiment, said hepatocytes are dog hepatocytes. It is not intendedthat the present invention be limited to any particular protein orpeptide; a variety are contemplated, including mixtures. For example, inone embodiment, the covalently attached protein is collagen. In anotherembodiment, a mixture of proteins are covalently attached, e.g. amixture of collagen type I, fibronectin and collagen type IV. In yetanother embodiment, the RGD peptide is attached (or a peptide comprisingthe RGD motif is attached). In one embodiment, the microchannel furthercomprises a membrane. In one embodiment, the membrane comprises PDMS. Inone embodiment, the membrane comprises a crosslinker (e.g. covalentlybound to the membrane). In one embodiment, the crosslinker is abifunctional crosslinker. In one embodiment, the crosslinker isSulfo-SANPAH (which is light activated with UV irradiation). In oneembodiment, an extracellular matrix protein (e.g. laminin) is attachedto the crosslinker (e.g. covalently bound) so as to provide anECM-coated membrane. In one embodiment, the irradiated membrane iswashed before the ECM attachment step. In one embodiment, the viablecells are further seeded onto the ECM-coated membrane. In oneembodiment, the viable cells are neurons. In one embodiment, the viablecells are motor neurons. In one embodiment, the viable cells arehepatocyte. In one embodiment, the viable cells are muscle cells. In oneembodiment, the viable cells are skeletal muscle cell. In oneembodiment, the skeletal muscle cells are human.

In one embodiment, the microchannel further comprises a micropatternedmembrane. In one embodiment, the micropatterned membrane comprises PDMS.In one embodiment, a bifunctional crosslinker is attached to themicropatterned membrane. In one embodiment, the micropatterned membraneis in the flow channel of a microfluidic device. In one embodiment, themicropattern is parallel to the fluid flow. In one embodiment, themicropattern is perpendicular to the fluid flow. In one embodiment, anextracellular matrix protein (e.g. laminin) is attached to thecrosslinker (e.g. covalently bound) so as to provide an ECM-coatedmicropatterned membrane. In one embodiment, the viable cells are furtherseeded onto the ECM-coated micropatterned membrane. In one embodiment,the viable cells are neurons. In one embodiment, the viable cells aremotor neurons. In one embodiment, the viable cells are hepatocyte. Inone embodiment, the viable cells are muscle cells. In one embodiment,the viable cells are skeletal muscle cell. In one embodiment, theskeletal muscle cells are human. In one embodiment, the skeletal musclecells elongate in the grooves of the micropatterned membrane.

In one embodiment, the crosslinker is only attached to a portion of themembrane or the micropatterned membrane. In one embodiment, the portionwhere the crosslinker is not attached is covered with a mask (e.g. thecrosslinker is light activated and the mask blocks the light) and theportion where the crosslinker is attached is unmasked. The mask may beadhesive material or non-adhesive material (e.g. metal or metal foilsuch as aluminum foil). In one embodiment, an extracellular matrixprotein (e.g. laminin) is attached to the crosslinker (e.g. covalentlybound) in the unmasked portion so as to provide an ECM-coated membraneor micropatterned membrane. In one embodiment, the viable cells arefurther seeded onto the ECM-coated portion of the membrane ormicropatterned membrane. In one embodiment, the viable cells areneurons. In one embodiment, the viable cells are motor neurons. In oneembodiment, the viable cells are hepatocyte. In one embodiment, theviable cells are muscle cells. In one embodiment, the viable cells areskeletal muscle cell. In one embodiment, the skeletal muscle cells arehuman.

It is not intended that the present invention be limited to the methodby which micropatterns are introduced into the membrane. In oneembodiment, the micropatterns are introduced into the membrane throughthe use of an existing micropatterned silicon wafer mold; PDMS materialcan be spin coated on the mold and cured (the membrane is thereafterremoved from the mold and used to assemble a microfluidic device orchip). In one embodiment, the micropattern is embossed using an existingmicropatterned silicon wafer using both heat and pressure.

It is not intended that the present invention be limited by the mannerin which the proteins or peptides are covalently attached. In oneembodiment, a crosslinker is used. In another embodiment, a bifunctionalcrosslinker is used. In a preferred embodiment, the protein or peptideis covalently attached to said microchannel surface singN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.

The present invention contemplates that the cells will be remain viableand can be tested to confirm this. However, it is not intended that thepresent invention be limited to a particular viability test. In oneembodiment, the method further comprises e) assessing viability bymeasuring the level of activity of one or more cellular enzymes. Avariety of enzymes can be used for this purpose, including but notlimited to, a CYP enzyme, a transaminase and the like. In anotherembodiment, the present invention contemplates that the method furthercomprises e) assessing viability by measuring the level of expression ofone or more cellular proteins.

The present invention contemplates that in certain embodiments, thesurface can be treated prior to step b). A variety of surface treatments(e.g. chemical vapor deposition, plasma oxidation, Corona, RF plasma,etc.) are possible. For example, in one embodiment, the presentinvention contemplates wherein said microchannel surface is PDMS andwherein said. PDMS is plasma treated prior to step b).

As noted above, cells need not be immediately cultured in the device,i.e. the device can be stored with the covalently attached protein(s).In one embodiment, the present invention contemplates a method oftreating a microfluidic device, comprising: a) providing a microfluidicdevice comprising a microchannel comprising a surface, said microchannelin fluidic communication with a fluid source comprising fluid; b)covalently attaching one or more proteins or peptides to saidmicrochannel surface so as to create a treated surface; and c) storingsaid microfluidic device. It is not intended that the present inventionbe limited to the precise storage conditions. However, the storing istypically done at a controlled temperature below room temperature, e.g.between 2 and 10° C., in a refrigerator or other cooling device. It isnot intended that the present invention be limited to dry or wetstorage. In one embodiment, said one or more covalently attachedproteins is collagen I and it is stored wet or dry (more preferred). Inone embodiment, said one or more covalently attached proteins is lamininand it is stored wet (preferred) or dry. Laminin and/or Matrigel arecontemplated for a variety of chips, including Intestine-on-Chip, BloodBrain Barrier (BBB)-on-Chip, and NeuroMuscular Junction (NMJ)-on-Chip.In one embodiment, the microchannel further comprises a membrane. In oneembodiment, the membrane comprises PDMS. In one embodiment, the membranecomprises a crosslinker (e.g. covalently bound to the membrane). In oneembodiment, the crosslinker is a bifunctional crosslinker. In oneembodiment, the crosslinker is Sulfo-SANPAH (which is light activatedwith UV irradiation). In one embodiment, an extracellular matrix protein(e.g. laminin) is attached to the crosslinker (e.g. covalently bound) soas to provide an ECM-coated membrane. In one embodiment, the irradiatedmembrane is washed before the ECM attachment step. In one embodiment,the viable cells are further seeded onto the ECM-coated membrane. In oneembodiment, the viable cells are neurons. In one embodiment, the viablecells are motor neurons. In one embodiment, the viable cells arehepatocyte. In one embodiment, the viable cells are skeletal musclecell. In one embodiment, the skeletal muscle cells are human.

In one embodiment, the microchannel further comprises a micropatternedmembrane. In one embodiment, the micropatterned membrane comprises PDMS.In one embodiment, a bifunctional crosslinker is attached to themicropatterned membrane. In one embodiment, the micropatterned membraneis in the flow channel of a microfluidic device. In one embodiment, themicropattern is parallel to the fluid flow. In one embodiment, themicropattern is perpendicular to the fluid flow. In one embodiment, anextracellular matrix protein (e.g. laminin) is attached to thecrosslinker (e.g. covalently bound) so as to provide an ECM-coatedmicropatterned membrane. In one embodiment, the viable cells are furtherseeded onto the ECM-coated micropatterned membrane. In one embodiment,the viable cells are neurons. In one embodiment, the viable cells aremotor neurons. In one embodiment, the viable cells are hepatocyte. Inone embodiment, the viable cells are muscle cells. In one embodiment,the viable cells are skeletal muscle cell. In one embodiment, theskeletal muscle cells are human. In one embodiment, the skeletal musclecells elongate in the grooves of the micropatterned membrane.

In one embodiment, the crosslinker is only attached to a portion of themembrane or the micropatterned membrane. In one embodiment, the portionwhere the crosslinker is not attached is covered with a mask (e.g. thecrosslinker is light activated and the mask blocks the light) and theportion where the crosslinker is attached is unmasked. The mask may beadhesive material or non-adhesive material (e.g. metal or metal foilsuch as aluminum foil). In one embodiment, an extracellular matrixprotein (e.g. laminin) is attached to the crosslinker (e.g. covalentlybound) in the unmasked portion so as to provide an ECM-coated membraneor micropatterned membrane. In one embodiment, the viable cells arefurther seeded onto the ECM-coated portion of the membrane ormicropatterned membrane. In one embodiment, the viable cells areneurons. In one embodiment, the viable cells are motor neurons. In oneembodiment, the viable cells are hepatocyte. In one embodiment, theviable cells are muscle cells. In one embodiment, the viable cells areskeletal muscle cell. In one embodiment, the skeletal muscle cells arehuman.

It is not intended that the present invention be limited to the methodby which micropatterns are introduced into the membrane. In oneembodiment, the micropatterns are introduced into the membrane throughthe use of an existing micropatterned silicon wafer mold; PDMS materialcan be spin coated on the mold and cured (the membrane is thereafterremoved from the mold and used to assemble a microfluidic device orchip). In one embodiment, the micropattern is embossed using an existingmicropatterned silicon wafer using both heat and pressure.

The storage may be for a time period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more days—and even may be done for 3, 4 or 5 weeks.After storage, it is contemplated that cells will be added. For example,in one embodiment, the present invention contemplates that the methodfurther comprises: d) seeding viable cells on said treated surface so asto create attached cells; e) flowing fluid from said fluid sourcethrough said microchannel so as to create flowing conditions; and f)culturing said attached cells under said flow conditions such that saidcells remain attached and viable (e.g. viable for at least 14 days).

Again, any type of cell (or combination of cells) can be seeded. Cellsmay be of any cell type from a multicellular structure, includingnematodes, amoebas, up to mammals such as humans. The cell types seededon the device may simply depend on the type of organ (lung, liver,intestine, brain, kidney etc.) or organ function one wishes to mimic,and the tissues that comprise those organs. One can also co-culturevarious stem cells, such as bone marrow cells, induced adult stem cells,embryonal stem cells or stem cells isolated from adult tissues. In oneembodiment said cells are hepatocytes. In one embodiment, said cells areHuman Umbilical Vein Endothelial Cells (HUVECs). In one embodiment, saidcells are intestinal cells. In one embodiment, said cells are frompatients with a disease, i.e. diseased cells. In one embodiment, saidcells are from healthy controls.

The present invention contemplates that in certain embodiments, thesurface can be treated prior to step b). A variety of surface treatments(e.g. chemical vapor deposition, plasma oxidation, Corona, RF plasma,etc.) are possible. For example, in one embodiment, the presentinvention contemplates wherein said microchannel surface is PDMS andwherein said PDMS is plasma treated prior to step b).

A noted above, the cells may be tested for viability. On the other hand,the cells may be put to other tests, such as tests directed at basicbiological science, life science research, drug discovery anddevelopment, drug safety testing, chemical and biological assays, aswell as tissue and organ engineering. In an embodiment, the organ mimicdevice can be used as microvascular network structures for basicresearch in cardiovascular, cancer, and organ-specific disease biology.Furthermore, one or more embodiments of the device find application inorgan assist devices for liver, kidney, lung, intestine, bone marrow,and other organs and tissues, as well as in organ replacementstructures.

It is not intended that the devices be limited in their use. In oneembodiment, there are used for: the identification of markers ofdisease; assessing efficacy of anti-cancer therapeutics; testing genetherapy vectors; drug development; screening; studies of cells,especially stem cells and bone marrow cells; studies onbiotransformation, absorption, clearance, metabolism, and activation ofxenobiotics; studies on bioavailability and transport of chemical orbiological agents across epithelial or endothelial layers; studies ontransport of biological or chemical agents across the blood-brainbarrier; studies on transport of biological or chemical agents acrossthe intestinal epithelial barrier; studies on acute basal toxicity ofchemical agents; studies on acute local or acute organ-specific toxicityof chemical agents; studies on chronic basal toxicity of chemicalagents; studies on chronic local or chronic organ-specific toxicity ofchemical agents; studies on teratogenicity of chemical agents; studieson genotoxicity, carcinogenicity, and mutagenicity of chemical agents;detection of infectious biological agents and biological weapons;detection of harmful chemical agents and chemical weapons; studies oninfectious diseases; studies on the efficacy of chemical or biologicalagents to treat disease; studies on the optimal dose range of agents totreat disease; prediction of the response of organs in vivo tobiological agents; prediction of the pharmacokinetics of chemical orbiological agents; prediction of the pharmacodynamics of chemical orbiological agents; studies concerning the impact of genetic content onresponse to agents; studies on gene transcription in response tochemical or biological agents; studies on protein expression in responseto chemical or biological agents; studies on changes in metabolism inresponse to chemical or biological agents. The organ mimic device canalso be used to screen on the cells, for an effect of the cells on thematerials (for example, in a manner equivalent to tissue metabolism of adrug).

In one embodiment, the present invention contemplates a method ofculturing cells, comprising: a) providing a microfluidic devicecomprising a microchannel comprising a surface, said microchannel influidic communication with a fluid source comprising fluid; b)covalently attaching a bifunctional crosslinker to said surface tocreate attached crosslinker, c) covalently attaching one or moreproteins or peptides to said attached crosslinker as to create a treatedsurface; d) seeding viable cells on said treated surface so as to createattached cells; e) flowing fluid from said fluid source through saidmicrochannel so as to create flowing conditions; and f) culturing saidattached cells under said flow conditions such that said cells remainattached and viable for at least 7 days. In one embodiment, said surfaceis a membrane and said membrane is micropatterned. In one embodiment,said cells are muscle cells that align with said micropattern (of themicropatterned membrane). In one embodiment, said crosslinker isactivated with UV light in the presence of a mask (so that light isblocked from contacting a portion of said surface).

In one embodiment, the present invention contemplates a method ofculturing cells, comprising: a) providing a microfluidic devicecomprising a surface; b) covalently attaching one or more proteins orpeptides to said surface using a crosslinker so as to create a treatedsurface; c) seeding viable cells on said treated surface so as to createattached cells; and d) culturing said attached cells such that saidcells remain attached and viable for at least 7 days. In one embodiment,the microfluidic device further comprises a microchannel, said surfacedisposed within said microchannel, and wherein said microchannel is influidic communication with a fluidic source comprising fluid, the methodfurther comprising the step of flowing fluid from said fluid sourcethrough said microchannel so as to create flow conditions, and whereinsaid culturing in d) further comprises culturing said attached cellsunder said flow conditions. In one embodiment, the attached cellsfurther remain viable for at least 14 days. In one embodiment, theattached cells further remain functional for at least 7 days. In oneembodiment, the attached cells further remain functional for at least 14days. In one embodiment, the crosslinker comprises at least onelight-reactive portion and at least one chemically reactive portion. Inone embodiment, the crosslinker further comprises at least one spacerportion. In one embodiment, the at least one light-reactive portion isselected from the group consisting of a nitrophenyl, a diazirine and anazide. In one embodiment, the at least one chemically reactive portionis selected from the group consisting of an NHS-ester, asulfo-NHS-ester, isocyanate, isothiocyanate, imidoester, maleimide,pyridyldithiol, and hydrazide. In one embodiment, the crosslinker isselected from the group consisting of sulfo-SANPAH, SANPAH, SDA,sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS, SDAD, sulfo-SDAD. In oneembodiment, the surface comprises PDMS. In one embodiment, the surfaceis plasma treated prior to step b). In one embodiment, the cells arehepatocytes. In one embodiment, the method further comprises step e)assessing viability by measuring the level of activity of one or morecellular enzymes. In one embodiment, the cellular enzyme is a CYPenzyme. In one embodiment, the cellular enzyme is a transaminase. In oneembodiment, the method further comprises step e) assessing viability bymeasuring the level of expression of one or more cellular proteins. Inone embodiment, the one or more proteins comprises collagen. In oneembodiment, the one or more proteins comprises a mixture of collagentype I, fibronectin and collagen type IV. In one embodiment, the one ormore peptides comprises RGD or a peptide comprising the RGD motif. Inone embodiment, wherein RGD is covalently attached to said surface usingN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate. In oneembodiment, the covalently attaching one or more proteins or peptides instep b) further comprises: i) introducing said crosslinker or a solutioncontaining said crosslinker to contact said surface and permitting saidcrosslinker or said solution containing said crosslinker to react withsaid surface; and ii) introducing at least one protein or peptide, or asolution containing at least one protein or peptide to contact saidsurface. In one embodiment, the covalently attaching one or moreproteins or peptides in b) further comprises exposing at least a portionof said surface to light. In one embodiment, the light comprises UVlight. In one embodiment, the exposing comprises exposing a selectedarea or pattern for the covalent attachment of at least a portion ofsaid one or more proteins or peptides. In one embodiment, the exposingcomprises masking said light so as to select said selected area orpattern. In one embodiment, the exposing comprises projecting a lightpattern so as to select said selected area or pattern. In oneembodiment, the exposing comprises rastering light so as to select saidselected area or pattern. In one embodiment, the selected area orpattern comprises a linear pattern. In one embodiment, the cellscomprise muscle cells or muscle-like cells that align with respect tosaid selected area of pattern. In one embodiment, the covalentlyattaching one or more proteins or peptides in b) further comprisesintroducing said crosslinker to contact only one or more selected areasof said microfluidic device. In one embodiment, the microfluidic devicefurther comprises a porous membrane. In one embodiment, the porousmembrane comprises said surface.

In one embodiment, the present invention contemplates a method oftreating a microfluidic device, comprising: a) providing a microfluidicdevice comprising a surface; b) covalently attaching one or moreproteins or peptides to said surface using a crosslinker so as to createa treated surface; and c) storing said microfluidic device. In oneembodiment, the microfluidic device comprises a microchannel, saidsurface disposed within said microchannel. In one embodiment, thestoring in step c) comprises storing said surface dry. In oneembodiment, the storing in step c) comprises storing said surface wet.In one embodiment, the crosslinker comprises at least one light-reactiveportion, at least one chemically reactive portion. In one embodiment,the crosslinker further comprises at least one spacer portion. In oneembodiment, the at least one light-reactive portion is selected from thegroup consisting of a nitrophenyl, a diazirine and an azides. In oneembodiment, the at least one chemically reactive portion is selectedfrom the group consisting of NHS-ester, sulfo-NHS-ester, isocyanate,isothiocyanate, imidoester, maleimide, pyridyldithiol, and hydrazide. Inone embodiment, the crosslinker is selected from the list comprising:sulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS,SDAD, sulfo-SDAD. In one embodiment, the storing is done at a controlledtemperature below room temperature. In one embodiment, the storing isdone at between 2 and 10° C. In one embodiment, the one or morecovalently attached proteins is collagen I. In one embodiment, thecovalently attached collagen I is stored dry. In one embodiment, the oneor more covalently attached proteins is laminin. In one embodiment, thecovalently attached laminin is stored wet. In one embodiment, the methodfurther comprises step d) seeding viable cells on said treated surfaceso as to create attached cells; and f) culturing said attached cellssuch that said cells remain attached and viable for at least 7 days. Inone embodiment, the microfluidic device further comprises amicrochannel, said surface disposed within said microchannel, andwherein said microchannel is in fluidic communication with a fluidicsource comprising fluid, the method further comprising flowing fluidfrom said fluid source through said microchannel so as to create flowconditions, and wherein culturing in f) further comprises culturing saidattached cells under said flow conditions. In one embodiment, theattached cells further remain viable for at least 14 days. In oneembodiment, the attached cells further remain functional for at least 7days. In one embodiment, the attached cells further remain functionalfor at least 14 days. In one embodiment, the attached cells arehepatocytes. In one embodiment, the surface comprises PDMS. In oneembodiment, the surface is plasma treated prior to step b). In oneembodiment, the covalently attaching one or more proteins or peptides inb) further comprises: i) introducing said crosslinker or a solutioncontaining said crosslinker to contact said surface and permitting saidcrosslinker or said solution containing said crosslinker to react withsaid surface; and ii) introducing at least one protein or peptide, or asolution containing at least one protein or peptide to contact saidsurface. In one embodiment, the covalently attaching one or moreproteins or peptides in b) further comprises exposing at least a portionof said surface to light. In one embodiment, the light comprises UVlight. In one embodiment, the exposing comprises exposing a selectedarea or pattern for the covalent attachment of at least a portion ofsaid one or more proteins or peptides. In one embodiment, the exposingcomprises masking said light so as to select said selected area orpattern. In one embodiment, the exposing comprises projecting a lightpattern so as to select said selected area or pattern. In oneembodiment, the exposing comprises rastering light so as to select saidselected area or pattern. In one embodiment, the selected area orpattern comprises a linear pattern. In one embodiment, the cellscomprise muscle cells or muscle-like cells that align with respect tosaid selected area or pattern. In one embodiment, the covalentlyattaching one or more proteins or peptides in b) further comprisesintroducing said crosslinker to contact one or more selected areas ofsaid microfluidic device. In one embodiment, the microfluidic devicefurther comprises a porous membrane. In one embodiment, the membranecomprises said surface.

In one embodiment, a method of culturing cells, comprising: a) providinga microfluidic device comprising a microchannel comprising a surface,said microchannel in fluidic communication with a fluid sourcecomprising fluid; b) covalently attaching a crosslinker to said surfaceto create attached crosslinker, c) covalently attaching one or moreproteins or peptides to said attached crosslinker as to create a treatedsurface; d) seeding viable cells on said treated surface so as to createattached cells; e) flowing fluid from said fluid source through saidmicrochannel so as to create flowing conditions; and f) culturing saidattached cells under said flow conditions such that said cells remainattached and viable for at least 7 days. In one embodiment, the surfaceis a membrane and said membrane is micropatterned. In one embodiment,the cells are muscle cells that align with said micropattern. In oneembodiment, the crosslinker is activated with UV light in the presenceof a mask.

In one embodiment, the present invention contemplates a kit comprising:a) a microfluidic device comprising a surface; b) a crosslinkercomprising at least one light-reactive portion, and at least onechemically reactive portion; c) at least one protein or peptide; and d)a set of instructions. In one embodiment, the kit further comprisescells.

In one embodiment, a method of culturing cells, comprising: a) providinga microfluidic device comprising a surface; b) covalently attaching oneor more proteins or peptides to said surface at a selected area orpattern using a crosslinker so as to create a treated surface; c)seeding viable cells on said treated surface so as to create attachedcells; and d) culturing said attached cells. In one embodiment, themicrofluidic device comprises a microchannel, said surface disposedwithin said microchannel, and wherein said microchannel is in fluidiccommunication with a fluidic source comprising fluid, the method furthercomprising flowing fluid from said fluid source through saidmicrochannel so as to create flow conditions, and wherein culturing ind) further comprises culturing said attached cells under said flowconditions. In one embodiment, the crosslinker comprises at least onelight-reactive portion, at least one chemically reactive portion. In oneembodiment, the crosslinker further comprises at least one spacerportion. In one embodiment, the at least one light-reactive portion isselected from the group consisting of a nitrophenyl, a diazirine and anazides. In one embodiment, the at least one chemically reactive portionis selected from the group consisting of NHS-ester, sulfo-NHS-ester,isocyanate, isothiocyanate, imidoester, maleimide, pyridyldithiol, andhydrazide. In one embodiment, the crosslinker is selected from the groupconsisting of sulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA,sulfo-LC-SDA, ANB-NOS, SDAD, and sulfo-SDAD. In one embodiment, thesurface comprises PDMS. In one embodiment, the surface is plasma treatedprior to step b). In one embodiment, the attached cells further remainviable for at least 7 days. In one embodiment, the attached cellsfurther remain functional for at least 7 days. In one embodiment, theattached cells further remain functional for at least 14 days. In oneembodiment, the method further comprises storing said microfluidicdevice before step c). In one embodiment, the covalently attaching oneor more proteins or peptides in b) further comprises: i) introducingsaid crosslinker or a solution containing said crosslinker to contactsaid surface and permitting said crosslinker or said solution containingsaid crosslinker to react with said surface; and ii) introducing atleast one protein or peptide, or a solution containing at least oneprotein or peptide to contact said surface. In one embodiment, thecovalently attaching one or more proteins or peptides in b) furthercomprises exposing at least a portion of said surface to light. In oneembodiment, the light comprises UV light. In one embodiment, theexposing comprises masking said light so as to select said selected areaor pattern. In one embodiment, the exposing comprises projecting a lightpattern so as to select said selected area or pattern. In oneembodiment, the exposing comprises mastering light so as to select saidselected area or pattern. In one embodiment, the selected area orpattern comprises a linear pattern. In one embodiment, the cellscomprise muscle cells or muscle-like cells that align with respect tosaid selected area or pattern. In one embodiment, the microfluidicdevice further comprises a porous membrane. In one embodiment, themembrane comprises said surface.

In one embodiment, the present invention contemplates a microfluidicdevice for culturing cells, comprising a) a surface; b) one or moreproteins or peptides attached to at least one portion of said surface bya crosslinker, said crosslinker comprising a light-reactive portion anda chemically reactive portion; wherein at least one chemical moiety ofsaid light-reactive portion is covalently attached to said surface, andat least one chemical moiety of said chemically reactive portion iscovalently attached to said one or more proteins or peptides. In oneembodiment, the device further comprises a microchannel, said surfacedisposed within said microchannel, and wherein said microchannel is influidic communication with a fluidic source. In one embodiment, the atleast one chemical moiety of said light-reactive portion is selectedfrom the group consisting of a reacted nitrophenyl, a reacted diazirineand a reacted azide. In one embodiment, the at least one chemical moietyof said chemically reactive portion is selected from the groupconsisting of a reacted NHS ester, a reacted sulfo-NHS-ester, a reactedisocyanate, a reacted isothiocyanate, a reacted imidoester, a reactedmaleimide, a reacted pyridyldithiol, and reacted hydrazide. In oneembodiment, the crosslinker is selected from the group consisting ofsulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS,SDAD and sulfo-SDAD. In one embodiment, the surface comprises PDMS. Inone embodiment, the at least one portion of said surface comprises aselected pattern. In one embodiment, the selected pattern comprises alinear pattern. In one embodiment, the device further comprises cellsdisposed in contact with said one or more proteins or peptides. In oneembodiment, the cells comprise muscle cells or muscle-like cells thatalign with respect to said selected pattern. In one embodiment, themicrofluidic device further comprises a porous membrane. In oneembodiment, the membrane comprises said at least one portion of saidsurface.

Definitions

The term “channels” refer to pathways (whether straight, curved, single,multiple, in a network, etc.) through a medium (e.g., silicon, glass,polymer, etc.) that allow for movement of liquids and gasses. Channelsthus can connect other components, i.e., keep components “incommunication” and more particularly, “in fluidic communication” andstill more particularly, “in liquid communication.” Such componentsinclude, but are not limited to, liquid-intake ports and gas vents.Microchannels are channels with dimensions less than 1 millimeter andgreater than 1 micron. It is not intended that the present invention belimited to only certain microchannel geometries. In one embodiment, afour-sided microchannel is contemplated. In another embodiment, themicrochannel is circular (in the manner of a tube) with curved walls. Inyet another embodiment, a combination of circular or straight walls isused.

The term “microfluidic” as used herein relates to components wheremoving fluid is constrained in or directed through one or more channelswherein one or more dimensions are 1 mm or smaller (microscale).Microfluidic channels may be larger than microscale in one or moredirections, though the channels) will be on the microscale in at leastone direction. In some instances the geometry of a microfluidic channelmay be configured to control the fluid flow rate through the channel(e.g. increase channel height to reduce shear). Microfluidic channelscan be formed of various geometries to facilitate a wide range of flowrates through the channels.

The term “arginylglycylaspartic acid” or “RGD” refers to a tripeptidecomposed of L-arginine, glycine and L-aspartic acid. RGD andRGD-peptides (i.e. peptides that are more than 3 amino acids in lengththat contain the RGD motif), such as GRGDSP, are implicated in cellularattachment via integrins.

The term “microfluidic device” refers to a substrate comprising at leastone channel that is configured to support fluid flow. Such a device maybe constructed out of a variety of materials including, but not limitedto, quartz, glass, plastic and/or PDMS or other polymer(s). For example,some microfluidic devices may comprise a microchip.

The term “seed” or “seeding” as used herein, refers to the attachmentand growth of cells on a surface of a microfluidic device, for example,within a channel or on a membrane of the microfluidic device.

The term “viable” as used herein, refers to any cell or group of cellsthat have demonstrated the capability of growing, dividing, developingand/or differentiating. Further, viability may be demonstrated by theidentification of specific biomarkers known the art for certain celltypes and/or organs.

The term, “surface” as used herein refers to any substrate as well assolid substrates which may comprise an array, microarray or microdevice.In some cases, the substrate is solid and may comprise PDMS.

The term, “muscle” as used herein refers to any group of cells or tissuehaving contractile capability including, but not limited to, skeletalmuscle, smooth muscle, cardiac muscle, myofibroblasts, pericytes, musclecells and muscle-like cells.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the chemical structure of the commercially availableheterobifunctional linkerN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate(Sulfa-SANPAH).

FIG. 2 shows the chemical surface modification to bind EMC protein tonative PDMS via linker molecule.

FIG. 3A-D show photographs of hepatocytes six (6) days after beingseeded on a PDMS surface that was either plasma treated (FIGS. 3 A & B)or that was Sulfo-SANPAH treated (i.e. ECM protein(s) covalentlyattached to the surface with this crosslinker) (FIGS. 3 C & D). Thecells were cultured under flow conditions for two (2) days.

FIG. 4A-D show photographs of hepatocytes nine (9) days after beingseeded on a PDMS surface that was either plasma treated (FIGS. 4A & B)or that was Sulfo-SANPAH treated (i.e. ECM protein(s) covalentlyattached to the surface with this crosslinker) (FIGS. 4C & D). The cellswere cultured under flow conditions for 5 days.

FIGS. 5 A&B show photographs of hepatocytes fourteen (14) days afterbeing seeded on a PDMS surface that was either plasma treated (FIG. 5A)or that was Sulfo-SANPAH treated (i.e. ECM protein(s) covalentlyattached to the surface with this crosslinker) (FIG. 5B). The cells werecultured under flow conditions for 10 days. ECM: Collagen type I 100μg/ml+FN 50 ug/ml+Collagen type IV 50 ug/ml. Cells on the Sulfo-SANPAHtreated surface (right) maintained monolayer over 14 days in culture.Cells on the plasma treated surface (left) started to detach (seearrow).

FIG. 6 is a drawing showing the extracellular matrix (ECM) next toprimary hepatocytes and endothelial cells.

FIG. 7A-C are photographs showing examples of liver cells (hepaticcells) on ECM coated chips under various conditions. Chips were coatedwith collagen I and fibronectin and stored either dry or wet for oneweek. Cells were then added to the chips and cultured for 14 days. As acontrol, a chip was coated fresh (no storage) and cultured with cellsfor 14 days. No differences in cell attachment were observed in Liversinusoidal endothelial cells (LSECs) or Hepatic cells (Hep). Nodifferences in morphology were observed (LSEC and Hep). FIG. 7A is thecontrol (fresh ECM coat) after 14 days of cell culture. FIG. 7B showsthe results after 1 week wet storage and cell culture for 14 days. FIG.7C shows the results after 1 week dry storage and cell culture for 14days.

FIGS. 8A&B are photographs showing the results from a one month storagestudy. Chips were coated with collagen I and fibronectin and stored dryfor one month. Liver cells were then added to the chips and cultured for13 days (FIG. 8B). As a control, a chip was coated fresh (no storage)and cultured with cells for 13 days (FIG. 8A). No differences in cellattachment were observed (LSEC and Hep). No differences in morphologywere observed (LSEC and Hep)

FIGS. 9A&B are bar graphs showing comparisons of different liver on chipexamples. Chips were coated with collagen I and fibronectin and storeddry for one month. Liver cells (Hepaptocytes) were then added to thechips and cultured (grey bars). As a control, a chip was coated fresh(no storage) and cultured with cells (blue bars). Albumin was measuredin the culture fluid after 6 and 13 days of culture (FIG. 9A). LDH wasmeasured in the culture fluid after 6 and 13 days (FIG. 9B).

FIG. 10A-C are photographs showing the results from a 1 week gut-on-chipstorage study. Chips were ECM coated (Matrigel and collagen I) andstored wet for 1 week. Thereafter, Human Umbilical Vein EndothelialCells (HUVEC) and Caco-2 cells were cultured on the chip for 11 days.Caco-2 cells and HUVEC were on each chip with the Caco-2 cells are onthe top side of the membrane and the HUVEC are on the bottom. FIG. 10A-Cimages were taken at the point of the chip where the two channels join,the wall of the channel is the dark separator in the images. This is atop-down view. The gut function is assessed via barrier function (pApp,the system's permeability coefficient) and response to stimulation(using an inflammatory stimulus).

FIGS. 11A&B are photographs showing small amounts of air observed inbottom channel inlets after wet storage because PDMS is vapor permeable.FIG. 11A shows wet storage of 3 weeks. FIG. 11B shows a close up of airobserved in the bottom channel inlets after wet storage for 4 weeks.Small amounts of air observed in bottom channel inlets after 12 days ofstorage. Air volume increased after 3 weeks (FIG. 11A). By 30 days, mostECM solution has evaporated from the chip.

FIG. 12 is a graph showing the increasing barrier function of cells inthe gut-on-chip. Chips were coated with Matrigel and collagen I andstored wet for one week. Thereafter, the gut cells Caco-2 and HUVECcells) were added to the chip and cultured. The results demonstratehealthy and functional cell populations.

FIG. 13A-C presents exemplary data showing a concentration dependenteffect of laminin binding subsequent to 500 μg/ml Sulfo-SANPAH (IV)treated channels.

FIG. 13A: 10 μg/ml Laminin treated channels.

FIG. 13B: 50 μg/ml Laminin treated channels.

FIG. 13C: 100 μg/ml Laminin treated channels.

FIGS. 14A&B presents exemplary data showing a concentration dependenteffect of laminin on the development and differentiation of motor neuroncells cultured on chips.

FIG. 14A: Motor neurons cultured in channels treated with 50 μg/mlLaminin.

FIG. 14B: Motor neurons cultured in channels treated with 100 μg/mlLaminin.

FIGS. 15A&B presents alternative embodiments of PDMS membranemicropatterning.

FIG. 15A: Micropatterning perpendicular to channel fluid flow.

FIG. 15B: Micropatterning parallel to channel fluid flow.

FIG. 16A-D presents exemplary data showing nuclei development withinhuman skeletal muscle cell (hSKMC) myotubes.

FIG. 16A: Phase contrast photomicrograph of skeletal muscle cellalignment along a micropatterned PDMS membrane.

FIG. 16B: Photomicrograph (10×) of hSKMC myotube nuclei development onDay 3 of culture on a micropatterned PDMS membrane.

FIG. 16C: Photomicrograph (10×) of hSKMC myotube nuclei development onDay 11 of culture on a micropatterned PDMS membrane.

FIG. 16D: Photomicrograph (20×) of hSKMC myotube nuclei development onDay 11 of culture on a micropatterned PDMS membrane.

FIG. 17A-D presents exemplary data showing actin growth and developmentwithin human skeletal muscle cell (hSKMC) myotubes.

FIG. 17A: Photomicrograph (10×) of actin development on anon-micropatterned PDMS membrane on culture Day 7.

FIG. 17B: Photomicrograph (20×) of actin development on anon-micropatterned PDMS membrane on culture Day 7.

FIG. 17C: Photomicrograph (10×) of actin development on a micropatternedPDMS membrane on culture Day 7.

FIG. 17D: Photomicrograph (20×) of actin development on a micropatternedPDMS membrane on culture Day 7.

FIG. 18 presents exemplary data in a bar graph showing hSKMC morphology(e.g., elongated or round) as measured by a cell shape index.

FIG. 19A and FIG. 19B show exemplary data showing greater hSKMCattachment to PDMS membranes in unmasked regions versus masked regionsdue to UV-activated crosslinkers.

FIG. 20A-C presents exemplary data showing the fabrication and use of anembossed PDMS membrane.

FIG. 20A: An embossed PDMS membrane before cell attachment.

FIG. 20B: An embossed PDMS membrane subsequent to one day of cellattachment and culture.

FIG. 20C: An embossed PDMS membrane subsequent to six days of cellattachment and culture.

FIG. 21 shows generic examples of potential crosslinkers with theformula A-B-C, wherein A represents light-reactive portion, B representsa linker, and C represents modifier-reactive portion. The formula on theleft represents a linear crosslinker, the formula in the center and theleft represent where the liken portion is multivalent.

FIG. 22 shows a specific example of a crosslinker, Sulfo-SANPAH which isdiagrammed according to the A-B-C crosslinker formula described above.

DETAILED DESCRIPTION OF THE INVENTION

Silicone elastomers, such as PDMS, are used in microfluidics. However,silicone polymers are hydrophobic and do not promote cell adhesion.Surface treatments (e.g. chemical vapor deposition, plasma oxidation,Corona, RF plasma, etc.) have been used to make such polymers moreuseful See e.g. Hong et al., “Hydrophilic Surface Modification of PDMSUsing Atmospheric RF Plasma,” Journal of Physics: conference Series 34(2006) 656-661 (Institute of Physics Publishing). A microfluidic device(or portion thereof) made of a naturally hydrophobic material becomeshydrophilic upon such surface treatment. Nonetheless, cell attachmentremains a problem, both in the short term and the long term. That is tosay, some cells do not adhere well to surface treated PDMS at theoutset; they exhibit low seeding levels. In the long term, cells (andeven monolayers) can detach from the surface treated PDMS.

The present invention contemplates compositions, devices and methods ofimproving adhesion, attachment, and/or differentiation of cells in amicrofluidic device or chip, and in particular, cells on a PDMS surface.In one embodiment, one or more proteins (e.g. ECM proteins) or peptides(e.g. RGD) are covalently coupled to the surface of a microchannel or amicrofluidic device. The microfluidic devices can be stored and laterused, or they can be immediately used for culture and/or support ofliving cells such as mammalian cells, and/or for simulating a functionof a tissue, e.g., a liver tissue, muscle tissue, etc. Even under flowconditions, extended adhesion and viability with sustained function overtime is observed.

The present invention contemplates microfluidic devices (or “chips”)containing living cells recreate the physiological tissue-tissueinterfaces and permit fluid flow. See U.S. Pat. No. 8,647,861, herebyincorporated by reference. Such devices subject the cells to shearstress. In contrast to static 2D culture, microchannels allow theperfusion of cell culture medium throughout the cell culture during invitro studies and as such offer a more in vivo-like physicalenvironment. In simple terms, an inlet port allows injection of fluidssuch as cell culture medium (and the like) into a microfluidic channelor chamber (with or without cells). In one embodiment, the presentinvention contemplates introduction of fluid into a cell-ladenmicrofluidic channel or chamber. In a preferred embodiment, the cellsare attached to one or more ECM proteins (e.g. laminin), which are inturn covalently attached to the microchannel surface. An outlet portthen permits the exit of remaining fluid as well as harmful metabolicby-products.

The surface over which the fluid flows and to which the cells areattached (using the methods described herein) can be a surface of anymaterial that is compatible to the fluid sample and cells. Exemplarymaterials for the fluid-contact surface can comprise glass, syntheticpolymers (e.g., PDMS, polysulfonate, and polycarbonate), hydrogels, anda combination thereof.

One portion of a microchannel can be a membrane. For example, the floorof a microchannel can comprise a membrane, including a porous membrane.The microchannel (or portion thereof) or membrane can be coated withsubstances such as various cell adhesion promoting substances or ECMproteins, such as fibronectin, laminin or various collagen types orcombinations thereof. For example, endothelial cells can attach to acollagen coated microchannel. While non-covalent coating can be used, itis preferred that such proteins and peptides be covalently attached,e.g. by use of a crosslinker or other chemistry.

It is not intended that the present invention be limited to the methodby which one or more ECM proteins are covalently attached to themicrochannel surface. In one embodiment, bifunctional crosslinkers areused. A variety of such crosslinkers are available commercially,including (but not limited to) the following compounds:

-   -   ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide) having the        formula of:

-   -   Sulfo-SAND (sulfosuccinimidyl        2-[m-azido-o-nitrobenzamido]ethyl-1, 3′-dithiopropionate) having        the formula of:

-   -   SANPAH        (N-succinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate)        having the formula of:

-   -   Sulfa-SANPAH        (sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate)        having the formula of:

By way of example, sulfosuccinimidyl 6-(4′-azide-2′-nitrophenyl-amino)hexanoate or “Sulfo-SANPAH” (commercially available from Pierce) is along-arm (18.2 angstrom) crosslinker that contains an amine reactiveN-hydroxysuccinimide (NHS) ester and a photoactivatable nitrophenylazide. NHS esters react efficiently with primary amino groups (—NH₂) inpH 7-9 buffers to form stable amide bonds. The reaction results in therelease of N-hydroxysuccinimide. When exposed to UV light, nitrophenylazides form a nitrene group that can initiate addition reactions withdouble bonds, insertion into C—H and N—H sites, or subsequent ringexpansion to react with a nucleophile (e.g., primary amines). The latterreaction path dominates when primary amines are present.

Sulfo-SANPAH should be used with non-amine-containing buffers at pH 7-9such as 20 mM sodium phosphate, 0.15M NaCl; 20 mM HEPES; 100 mMcarbonate/bicarbonate; or 50 mM borate. Tris, glycine orsulfhydryl-containing buffers should not be used. Tris and glycine willcompete with the intended reaction and thiols can reduce the azidogroup.

The present invention is not to be limited to any particularcrosslinker. In one embodiment, the crosslinkers of the currentinvention comprise three parts: a light-reactive portion, a linker, anda modifier-reactive portion. In one embodiment, the bifunctionalcrosslinkers are represented by the formula A-B-C, wherein A representslight-reactive portion, B represents a linker, and C representsmodifier-reactive portion. The present invention is not to be limited tolinear crosslinkers. In one embodiment, B can also be branched itmultivalent. i.e. it can link one A to two Cs, 3As to 4Cs, etc, see FIG.13. As a non-limiting example, sulfo-SANPAH uses a nitrophenyl azidegroup as the light-reactive portion, aminohexanoate as the linker, andsulfo-NHS ester as the modifier-reactive portion (in this case reactingwith an amine group on the modifier), see FIGS. 14A&B. In oneembodiment, light reactive portions may be selected from the groupconsisting of nitrophenyl, diazirine, and azides. The present inventionis not to be limited to any particular linker. In one embodiment, thelinker (B) are connected to light-reactive portion (A) through an aminebond and modifier-reactive portion (C) through an ester bond. In oneembodiment, the linkers may be selected from the group consisting ofpolyethyleneglycols, alkanes, and olefins. In one embodiment, themodifier-reactive chemistry portion may be selected from the groupconsisting of NHS-ester (amine reactive), Sulfo-NHS-ester (aminereactive), Isocyanate (amine reactive), Isothiocyanate (amine reactive),Imidoester (amine reactive), Maleimide (sulfhydryl reactive),Pyridyldithiol (sulfhydryl reactive), and Hydrazide (aldehyde and ketonereactive). Specific examples of commercially available crosslinkers thatfit this description include ANB-NOS, SDA, sulfo-SDA, LC-SDA,sulfo-LC-SDA, SDAD, sulfo-SDAD, and more (see Table 1). ANB-NOS is ashort-arm (7.7 angstrom) crosslinker that contains an amine-reactiveN-hydroxysuccinimide (NHS) ester and a photoactivatable nitrophenylazide, also called N-5-azido-2-nitrobenzoyloxysuccinimide. Sulfo-SANPAHis a long-arm (18.2 angstrom) crosslinker that contains anamine-reactive N-hydroxysuccinimide (NHS) ester and a photoactivatablenitrophenyl azide, also called sulfosuccinimidyl6-(4′-azido-2′-nitrophenylamino)hexanoate. SDA (NHS-Diazirine) combinesproven NHS-ester and diazirine-based photoreaction chemistries withconjugate amine-containing molecules with nearly any other functionalgroup via long-wave UV-light activation. SDA (Sulfo-NHS-Diazirine) is anamine and photoreactive, membrane impermeable, heterobifunctionalcrosslinker with a 3.9 Angstrom spacer arm. Also calledSulfosuccinimidyl 4,4′-azipentanoate. LC-SDA (NHS-LC-Diazirine) is anamine and photoreactive, membrane permeable, heterobifunctionalcrosslinker with a 12.5 Angstrom spacer arm. Also called Succinimidyl6-(4,4′-azipentanamido)hexanoate, Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine)is a sulfo-NHS-diazirine based photoreactive crosslinker. Membraneimpermeable with a 12.5 Angstrom spacer arm. Also calledSulfosuccinimidyl 6-(4,4′-azipentanamido)hexanoate.

TABLE 1 Examples of commercially available crosslinkers Reactive SpacerCleavable Membrane Groups Products Arm (Å) by? Water-soluble? permeable?NHS ester/ ANB-NOS 7.7 Short No No No aryl azide Sulfo-SANPAH 18.2 NoYes No Long NHS ester/ SDA 3.9 Short No No Yes diazirine Sulfo-SDA 3.9Short No Yes No LC-SDA 12.5 Mid No No Yes Sulfo-LC-SDA 12.5 Mid No YesNo SDAD 13.5 Mid Thiols No Yes Sulfo-SDAD 13.5 Mid Thiols Yes NoBy way of example, sulfosuccinimidyl 6-(4′-azido-2′-nitrophenyl-amino)hexanoate or “Sulfo-SANPAH” (commercially available from Pierce) is along-arm (18.2 angstrom) crosslinker that contains an amine-reactiveN-hydroxysuccinimide (NHS) ester and a photoactivatable nitrophenylazide, NHS esters react efficiently with primary amino groups (—NH2) inpH 7-9 buffers to form stable amide bonds. The reaction results in therelease of N-hydroxysuccinimide. When exposed to UV light, nitrophenylazides form a nitrene group that can initiate addition reactions withdouble bonds, insertion into C-H and N-H sites, or subsequent ringexpansion to react with a nucleophile (e.g., primary amines). The latterreaction path dominates when primary amines are present.

Sulfo-SANPAH should be used with non-amine-containing buffers at pH 7-9such as 20 mM sodium phosphate, 0.15M NaCl; 20 mM HEPES; 100 mMcarbonate/bicarbonate; or 50 mM borate. Tris, glycine orsulfhydryl-containing buffers should not be used. Tris and glycine willcompete with the intended reaction and thiols can reduce the azidogroup.

For photolysis, one should use a UV lamp that irradiates at 300-460 nm.High wattage lamps are more effective and require shorter exposure timesthan low wattage lamps. UV lamps that emit light at 254 nm should beavoided; this wavelength causes proteins to photodestruct. Filters thatremove light at wavelengths below 300 nm are ideal. Using a secondfilter that removes wavelengths above 370 nm could be beneficial but isnot essential.

Calfskin type I collagen has been covalently attached to apolyacrylamide surface using sulfo-SANPAH. See Gaudet, C., “Influence oftype I collagen surface density on Fibroblast Spreading, Motility, andContractility” Biophys J. 85(5): 3329-3335 (2003). Collagen I wascoupled to other surfaces using Sulfo-SANPAH in order to avoid potentialdifferences in ECM remodeling on different substrates. See Trappman etal. “Extracellular-matrix tethering regulates stem-cell fate,” NatureMaterials (2012) (on-line publication). RGD has been covalently attachedto a PDMS surface using sulfo-SANPAH. See Li et al., “RGDpeptide-conjugated poly(dimethylsiloxane) promotes adhesion,proliferation, and collagen secretion of human fibroblasts,” J. BiomedMat Res A. 79(4):989-98 (2006).

It is not intended that the present invention be limited by the numberor nature of channels in the microfluidic device. In some embodiments,the surface can be a surface of a fluid-flowing conduit or passagewaydisposed in a solid substrate. In some embodiments, the surface can be asolid surface. For example, in one embodiment, the solid surface can bea wall surface of a fluid channel, e.g., a microfluidic channel.

In one embodiment, the present invention contemplates a co-culture ofliver sinusoidal endothelial cells in one chamber with hepatocytes inother chamber(s) to establish hepatic function in vitro. In oneembodiment, the chambers are first and second microchannels aligned(e.g., vertically) with each other with one or more membranes separatingthem from each other (“liver-on-a-chip”). The liver-on-a-chip deviceshave been developed and optimized based on the basic design of anorgan-on-a-chip as described in the U.S. Pat. No. 8,647,861, and theInternational Patent App. No. PCT/US2014/071611, the contents of each ofwhich are incorporated herein by reference in their entireties. In someaspects, the inventors have optimized the design of the liver-on-a chipdevices and culture conditions to provide long-term hepatic culture withphysiologically relevant hepatic function (e.g., albumin and/or ureasecretion, and/or CYP 450 metabolic capacity) for different animalmodels, e.g., human, rats, and dogs.

In a preferred embodiment, the present invention contemplates amicrofluidic device comprising a microchannel, said microchannelcomprising a monolayer of viable hepatocytes adhered to a coating, saidcoating comprising at least one extracellular matrix protein covalentlycoupled to a microchannel surface. The viable hepatocytes can be derivedfrom different mammalian sources, including, e.g., but not limited tohumans, rats, mice, and dogs.

In one embodiment, the present invention contemplates covalentlyattaching one or more proteins or peptides to a surface in themicrofluidic device (e.g. to the membrane and/or one or moremicrochannel), and storing the microfluidic device for a week or more (amonth or more) prior to attaching cells. In one embodiment, the presentinvention contemplates dry storage.

Experimental results have shown that chips for liver samples may bestored dry. That is to say, the extracellular matrix protein can beattached to the chip and can be stored dry (prior to any cell culture).However, not all ECM proteins can be stored dry; empirically, it wasfound that only some ECMs can be stored dry (collagens, fibronectin(FN)). Chips were stored at 4° C. and then compared to freshly coatedchips.

FIG. 7A-C are photographs showing examples of liver cells (hepaticcells) on ECM coated chips under various conditions. Chips were coatedwith collagen I and fibronectin and stored either dry or wet for oneweek. Cells were then added to the chips and cultured for 14 days. As acontrol, a chip was coated fresh (no storage) and cultured with cellsfor 14 days. No differences in cell attachment were observed in Liversinusoidal endothelial cells (LSECs) or Hepatic cells (Hep). Nodifferences in morphology were observed (LSEC and Hep). FIG. 7A is thecontrol (fresh ECM coat) after 14 days of cell culture. FIG. 7B showsthe results after 1 week wet storage and cell culture for 14 days. FIG.7C shows the results after 1 week dry storage and cell culture for 14days.

FIGS. 8A&B are photographs showing the results from a one month storagestudy. Chips were coated with collagen I and fibronectin and stored dryfor one month. Liver cells were then added to the chips and cultured for13 days (FIG. 8B). As a control, a chip was coated fresh (no storage)and cultured with cells for 13 days (FIG. 8A). No differences in cellattachment were observed (LSEC and Hep). No differences in morphologywere observed (LSEC and Hep).

There was also an evaluation of biomarkers in order to compare betweenfreshly coated (control) chips and chips dry-stored for 1 month with 5chips tested per condition with liver. FIGS. 9A&B are bar graphs showingcomparisons of different liver on chip examples. Chips were coated withcollagen I and fibronectin and stored dry for one month. Liver cells(Hepaptocytes) were then added to the chips and cultured (second bars).As a control, a chip was coated fresh (no storage) and cultured withcells (first bars). Albumin was measured in the culture fluid after 6and 13 days of culture (FIG. 9A). LDH was measured in the culture fluidafter 6 and 13 days (FIG. 9B). Chips stored dry for 1 month showedhigher albumin production and lower LHD release over a 2-weekobservation period. The LDH release is not desirable. Therefore, drystorage of colI/FN-coated chips is a viable platform for use with thehuman Liver-on-Chip.

The above results indicate that ECMs for Liver-on-Chip can be storeddry. Chips can be coated with collagen I and Fibronectin and put in a 1week dry storage, and even a 1 month dry storage.

In one embodiment, the present invention contemplates covalentlyattaching one or more proteins or peptides to a surface in themicrofluidic device (e.g. to the membrane and/or one or moremicrochannel), and storing the microfluidic device for a week or more (amonth or more) prior to attaching cells. In one embodiment, the presentinvention contemplates wet storage. In one embodiment, the presentinvention contemplates vapor proof packaging.

In one embodiment, the present invention contemplates a membranecomprising a pattern. In one embodiment, the pattern is a line andgroove pattern. Although it is not necessary to understand the mechanismof an invention, it is believed that a line and groove pattern providesalignment for cells such as muscle cells. It is further believed that itis microgrooves on the surface of the membrane that guides such cellalignment. In one embodiment, the membrane is a PDMS membrane.

In a preliminary experiment, PDMS membrane grooves were made using anexisting micropatterned silicon wafer (e.g., 10 μm×10 μm×2 μm) as amold. In one embodiment, a bifunctional crosslinker is attached to themicropatterned membrane. In one embodiment, the micropatterned membraneis in the flow channel of a microfluidic device. In one embodiment, themicropattern is parallel to the fluid flow. In one embodiment, themicropattern is perpendicular to the fluid flow. In one embodiment, anextracellular matrix protein (e.g. laminin) is attached to thecrosslinker (e.g. covalently bound) so as to provide an ECM-coatedmicropatterned membrane. Human primary muscle cells were then culturedon the micropatterned PDMS membrane in a static culture condition. Cellalignment was measured by F-actin stain and image analysis. Whenmicropatterned membranes were fabricated according to the methodsdescribed herein, cell alignment measurements demonstrated cellelongation. See, FIGS. 17A-D and FIG. 18.

In one embodiment, the PDMS membrane or micropatterned membrane may beselectively coated with a crosslinker using a mask. In one embodiment,the present invention contemplates the crosslinker used is Sulfo-SANPAHand the masking is done to control surface coating of this lightactivated crosslinker in a closed chip system (i.e. UV light can be usedwithout opening the chip). For example, Sulfo-SANPAH may be applied viachannels and ultraviolet light can be shined over the chip with a mask(to block the light from striking a specific portion or portions of thechip) covering a portion of an area, or alternatively a patterned maskto create a surface pattern. In one embodiment, the mask is layered,deposited or simply positioned on top of a portion of the membrane andsubsequently exposed to UV light. Once the mask is removed, an ECMprotein (or proteins) can be attached to the bifunctional crosslinker.In one embodiment, the irradiated membrane is washed before the ECMattachment step. Thereafter, cells may be attached to the resultantpattern generated by the mask and cultured in a static condition.Although it is not necessary to understand the mechanism of aninvention, it is believed that most cells attach to ultravioletlight-exposed membrane areas (where the crosslinker was activated tobind) and very few cells attach in the masked areas (i.e., where therewas no ultraviolet light exposure).

In one embodiment, the membrane micropatterning may be achieved byembossing. A preliminary experiment was performed using apre-manufactured thin membrane layered on top of a conventionalmicropatterned silicon wafer which was then exposed to a high heat(e.g., about 80-90° C.) and a weight (to create pressure) for 24-48 hrsto create the embossed pattern. Subsequent to the embossing, themicropatterned membrane was removed from the silicon wafer. In oneembodiment, the micropatterned membrane comprises PDMS. In oneembodiment, the micropatterned membrane is in the flow channel of amicrofluidic device. In one embodiment, the micropattern is parallel tothe fluid flow. In one embodiment, the micropattern is perpendicular tothe fluid flow. In one embodiment, a bifunctional crosslinker isattached to the micropatterned membrane. In one embodiment, anextracellular matrix protein (e.g. laminin) is attached to thecrosslinker (e.g. covalently bound) so as to provide an ECM-coatedmicropatterned membrane. In one embodiment, the viable cells are furtherseeded onto the ECM-coated micropatterned membrane. In one embodiment,human primary muscle cells were attached to the membrane and cultured ina static condition. The data showed that the hSKMCs were bound to themembrane and observed to grow along the micropatterned grooves. Afterapproximately six days of culture the hSKMCs were aligned along themicrogrooves.

EXPERIMENTAL

The following are examples that further illustrate embodimentscontemplated by the present invention. It is not intended that theseexamples provide any limitations on the present invention.

Example 1 Cellular Crosslinking to Improve Cell Attachment to Channels

In one embodiment, the present invention contemplates using acrosslinker to covalently attach proteins or peptides that enhance cellattachment. In this example, a protocol for using Sulfa-SANPAH as thecrosslinker is provided as one embodiment of a method.

First, fresh 0.5 mM Sulfo-SANPAH (492.4 g/mol) solution in 50 mM HEPES(0.22 um sterile filtered, pH 7.4) (protect from light) is prepared.Then, an ECM solution is prepared (e.g. 50 ug/mL Laminin in PBS or mediawithout FBS) on ice.

The microfluidic device (“chip”) comprising a microchannel is thenplasma treated. Plasma—15 sccm O₂, 60 sec, 100 W.

The channels are then washed with 200 uL of 50 mM HEPES. Excess 50 mMHEPES is removed from the channel.

Sulfo-SANPAH is introduced into the microchannel by inserting a pipettip reservoir in a port of the chip. 100 uL of Sulfo-SANPAH solution isadded to the top channel, ejecting tip into inlet port. 50 uL ofSulfo-SANPAH solution is added to the bottom channel, ejecting tip intoinlet port. The channels are then inspected to be sure no bubbles arepresent. At this point, one carefully removes and discards the pipettips reservoirs without spilling excess reagent on surface of chip.

At this point, the chips are ready for light treatment. The chips areincubated in the UV lamp chamber. Decrease distance to UV lamp and chipswith tip boxes or lab-jack. The incubation is for 20 min at 0.72joules/cm². After UV treatment, Sulfo-SANPAH is removed from thechannels and each channel is washed twice with 200 uL 50 mM HEPES. Thechannel dried by removing or aspirating remaining HEPES buffer.

At this point, a protein or peptide can be attached. A solutioncontaining a protein (e.g. ECM solution) or peptide (to enhance cellbinding) can be loaded into the channels with one of the followingconditions: Overnight at 4° C. or Minimum of 1.5 hours at 37° C.

Before seeding with cells, the solution (e.g. ECM solution) should beremoved from the channels and the channels washed with 200 uL of desiredmedia or PBS.

Example 2 Channel Surface Modification to Improve Cell Attachment

In this example, PDMS surfaces treated with plasma were compared withPDMS surfaces modified by covalent attachment ECM proteins. FIGS. 3A-Bshow photographs of hepatocytes six (6) days after being seeded on aPDMS surface that was either plasma treated (FIGS. 3 A & B) or that wasSulfo-SANPAH treated (i.e. ECM protein(s) covalently attached to thesurface with this crosslinker) (FIGS. 3 C & D). The cells were culturedunder flow conditions for two (2) days.

FIGS. 4A-D show photographs of hepatocytes nine (9) days after beingseeded on a PDMS surface that was either plasma treated (FIGS. 4A & B)or that was Sulfo-SANPAH treated (i.e. ECM protein(s) covalentlyattached to the surface with this crosslinker) (FIGS. 4C & D). The cellswere cultured under flow conditions for 5 days.

FIGS. 5A-B show photographs of hepatocytes fourteen (14) days afterbeing seeded on a PDMS surface that was either plasma treated (FIG. 5A)or that was Sulfo-SANPAH treated (i.e. ECM protein(s) covalentlyattached to the surface with this crosslinker) (FIG. 5B). The cells werecultured under flow conditions for 10 days. ECM Collagen type I 100ug/ml FN 50 ug/ml+Collagen type IV 50 ug/ml. Cells on the Sulfo-SANPAHtreated surface (right) maintained monolayer over 14 days in culture.Cells on the plasma treated surface (left) started to detach (seearrow). Clearly, the Sulfo-SANPAH treatment was an improvement overplasma treatment.

Example 3 Shelf-Life Study of ECM-Coated Chips

This example evaluates conditions to avoid ECM (i.e., for example,laminin, Matrigel) inactivation found during dry storage. All testedchips were stored at 4′C then compared to freshly coated chips. Resultsindicate that ECM for Gut-on-Chip chips are best stored in solution(e.g., wet).

Experimental Design

The ECM for Gut-on Chip chips comprised Matrigel and collagen I.Gut-on-Chip chips were chosen as test platform due to its robustness tovarying culture conditions. In particular, test Chips were treated withSulfo-SANPAH and ECM (100 ug/mL Martigel and 25 ug/mL collagen I). Allconditions were compared to freshly coated chips.

Results

Twenty-eight (28 chips) were stored for 1 week. No differences in Caco-2and HUVEC cell attachment was observed. No differences in Caco-2 andHUVEC cell morphology was observed. The chips were maintained for 8 daysprior to exposure to TNF-α and IL-1β. The experiment also included somechips with lamina propria cells. Samples for barrier function andlactate dehydrogenase (LDH) were collected, along with cytokineprofiles.

It appears that a 1 week storage does not impact cell attachment ormorphology in the Gut-on-Chip configuration. FIG. 10A-C show photographsdemonstrating the results from a 1 week gut-on-chip storage study. Chipswere ECM coated (Matrigel and collagen I) and stored wet for 1 week.Thereafter, Human Umbilical Vein Endothelial Cells (HUVEC) and Caco-2cells were cultured on the chip for 11 days. Caco-2 cells and HUVEC wereon each chip with the Caco-2 cells on the top side of the membrane andthe HUVEC on the bottom side of the membrane. FIG. 10A-C show examplesof gut on chips where the chips have been stored for 1 week. FIG. 10A-Cimages were taken at the point of the chip where the two channels join,the wall of the channel is the dark separator in the images. This is atop-down view. The gut function is assessed via barrier function (pApp,the system's permeability coefficient) and response to stimulation(using an inflammatory stimulus). The increasing barrier function (seeFIG. 12) demonstrates healthy and functional cell populations andcorrelates with previous data. Caco-2 and HUVEC cells were used. Wetstorage (1 week) of Matrigel/ColI coated chips are a viable platform foruse with the human Gut-on-Chip.

FIG. 12 shows the increasing barrier function which demonstrates healthyand functional cell populations. The FIG. 12 graph shows the increasingbarrier function of cells in the gut-on-chip. Chips were coated withMatrigel and collagen I and stored wet for one week. Thereafter, the gutcells (Caco-2 and HUVEC cells) were added to the chip and cultured. Theresults demonstrate healthy and functional cell populations.

In conclusion, the data shows that the storage of Gut-on-Chip coatedwith extracellular protein should be stored wet, such that more than 1week leads to evaporation issues. However, with a vapor proof packaging,the wet storage lifespan would be expected to improve.

The present invention contemplates other chips, such as those that useLaminin and/or Matrigel and may include chips that could be used asIntestine-on-Chip, Blood Brain Barrier (BBB)-on-Chip, and neuromuscularjunction (NW-on-Chip.

Example 4 Enhanced Cell Attachment in Presence of Crosslinker andLaminin

This example shows that the coating of a channel with crosslinker andlaminin improves the binding of cells to chip channels. The experimentaldesign coated chip channels with one of several concentrations (0.1mg/ml, 0.5 mg/ml and 1 mg/ml) of Sulfo-SANPAH (IV), followed by one ofseveral concentrations (10 μg/ml, 50 mg/ml and 100 μg/ml) offluorescently labeled laminin (red).

In general the protocol was as follows:

-   -   1. Flush the chip with 70% ethanol briefly and wash with 50 mM        HEPES buffer.    -   2. Add 50 μl of Sulfo-SANPAH (IV) and incubate under an        ultraviolet light for approximately 20 min.    -   3. Wash with 200 μl of 50 mM HEPES buffer three (3) times.    -   4. Wash with 200 μl of DPBS twice.    -   5. Add 50 μl of Laminin and incubate at 4° C. overnight.    -   6. Next day, transfer the chip at 37° C. for at least 1 h.    -   7. Seed the chip with motor neuron cells and culture.        Data collected before seeding with motor neuron cells shows        increased laminin binding as the laminin concentration        increases. See, FIG. 13A-C, Data collected after seeding with        motor neuron cells shows that the cells grown on higher        concentrations of laminin were differentiated and developed        thicker axons. See, FIGS. 14A&B.

Example 5 Micropatterning of a Chip Membrane

This example shows one method of micropatterning a PDMS membrane tofacilitate cell alignment a chip. In general the protocol was asfollows:

-   -   1. Prepare silicon wafer mold comprising 10×2 μm grooves.    -   2. Spin coat PDMS on top of the silicone mold.    -   3. Cure at 60° C. for over 6 hours.    -   4. Delaminate the micropatterned PDMS membrane from the silicon        wafer mold and assemble the micropattern PDMS membrane into a        channel chip in either a perpendicular or parallel orientation        relative to fluid flow. See, FIG. 15A and FIG. 15B,        respectively.    -   5. Surface treat with Sulfo-SANPAH and coat chips with Laminin.    -   6. Seed human skeletal muscle cells (hSKMC)    -   7. Subsequent to culturing, observe cell morphology and cell        alignment relative to the micropatterning.    -   8. Stain for muscle markers to show viability, differentiation        and development.    -   9. Assess the hSKMCs for spontaneous contraction, cholinergic        stimulation and IHC

The data show: i) hSKMC alignment within the PDMS groove micropattern(FIG. 16A); ii) progressive nuclei development within hSKMC myotubesbetween Day 3 Day 11 of culture (FIGS. 16B-C); iii) greater actindevelopment within hSKMCs cultured on micropatterned membranes by Day 7(FIGS. 17A-D); iv) greater proportion of elongated versus round hSKMCmorphology by Day 7 (FIG. 18).

Example 6 Membrane Micropatterning Using a Mask

This example creates a membrane surface pattern using Sulfo-SANPH and amask.

In general the protocol was as follows:

-   -   1. Sulfa-SANPAH (0.5 mg/ml) was added onto PDMS membrane.    -   2. A mask (e.g. a piece of aluminum foil) was overlayed on top        of a portion of the PDMS membrane.    -   3. The membrane with mask was transferred under an ultraviolet        light and illuminated for 20 min    -   4. The membrane was washed and Laminin was added and incubated        for 2 h at 37° C. incubator    -   5. Next day, hSKMC cells w ere seeded on to the membrane    -   6. The seeded hSKMC cells were then cultured for 7 days.

The data show that the regions where Sulfur-SANPAH was activated byexposure to ultraviolet light have greater sSKMC cell attachment thanthose masked regions (e.g., no Sulfo-SANPAH activation). See, FIGS.19A&B.

Example 7 Membrane Micropatterning Using Embossing

This example creates a membrane surface pattern using embossing withheat a pressure. In general the protocol was as follows:

-   -   1. A PDMS membrane was embossed onto silicon wafer at 80° C. for        2 days with pressure.    -   2. At day 2, the embossed PDMS membrane was delaminated from the        wafer.    -   3. Sulfur-SANPAH (0.5 mg/ml) was added onto PDMS membrane and        treated under ultraviolet light for 20 min.    -   4. The irradiated membrane was washed and Laminin was added and        incubated for 2 h at 37° C.    -   5. Next day, hSKMC cells were seeded onto the membrane.    -   6. The seeded hSKMC cells were then cultured for 7 days

A representative embossed PDMS membrane patterns is shown in FIG. 20A.The growth of hSKMC cells was observed on the embossed PDMS membranepatterns after Day I and Day 6 of culture. See, FIG. 20B and FIG. 20C,respectively.

While embodiments and applications have been shown and described, itwould be apparent to those skilled in the art having the benefit of thisdisclosure that many more modifications than mentioned above arepossible without departing from the inventive concepts disclosed herein.It is specifically contemplated that some of the features describedabove can be combined. For example, the membrane can be micropatternedto create a first pattern (e.g. by molding or embossing features such aslines, grooves, etc.) and then a crosslinker can be used with a mask tocreate a second pattern. The embodiment(s), therefore, are not to berestricted except in the spirit of the appended claims.

We claim:
 1. A method of culturing cells, comprising: a) providing amicrofluidic device comprising a surface; b) covalently attaching one ormore proteins or peptides to said surface using a crosslinker so as tocreate a treated surface; c) seeding viable cells on said treatedsurface so as to create attached cells; and d) culturing said attachedcells such that said cells remain attached and viable for at least 7days.
 2. The method of claim 1, wherein said microfluidic device furthercomprising a microchannel, said surface disposed within saidmicrochannel, and wherein said microchannel is in fluidic communicationwith a fluidic source comprising fluid, the method further comprisingthe step of flowing fluid from said fluid source through saidmicrochannel so as to create flow conditions, and wherein said culturingin d) further comprises culturing said attached cells under said flowconditions.
 3. The method of claim 1, wherein said attached cellsfurther remain viable for at least 14 days.
 4. The method of claim 1,wherein said attached cells further remain functional for at least 7days.
 5. The method of claim 1, wherein said attached cells furtherremain functional for at least 14 days.
 6. The method of claim 1,wherein said crosslinker comprises at least one light-reactive portionand at least one chemically reactive portion.
 7. The method of claim 1,wherein said crosslinker further comprises at least one spacer portion.8. The method of claim 6, wherein said at least one light-reactiveportion is selected from the group consisting of a nitrophenyl, adiazirine and an azide.
 9. The method of claim 6, wherein said at leastone chemically reactive portion is selected from the group consisting ofan NHS-ester, a sulfo-NHS-ester, isocyanate, isothiocyanate, imidoester,maleimide, pyridyldithiol, and hydrazide.
 10. The method of claim 1,wherein said crosslinker is selected from the group consisting ofsulfo-SANPAH, SANPAH, SDA, sulfo-SDA, LC-SDA, sulfo-LC-SDA, ANB-NOS,SDAD, sulfo-SDAD.
 11. The method of claim 1, wherein said surfacecomprises PDMS.
 12. The method of claim 1, wherein said surface isplasma treated prior to step b).
 13. The method of claim 1, wherein saidcells are hepatocytes.
 14. The method of claim 1, further comprisingstep e) assessing viability by measuring the level of activity of one ormore cellular enzymes.
 15. The method of claim 14, wherein said cellularenzyme is a CYP enzyme.
 16. The method of claim 14, wherein saidcellular enzyme is a transaminase.
 17. The method of claim 1, furthercomprising step e) assessing viability by measuring the level ofexpression of one or more cellular proteins.
 18. The method of claim 1,wherein said one or more proteins comprises collagen.
 19. The method ofclaim 1, wherein said one or more proteins comprises a mixture ofcollagen type I, fibronectin and collagen type IV.
 20. The method ofclaim 1, wherein said one or more peptides comprises RGD or a peptidecomprising the RGD motif.
 21. The method of claim 20, wherein RGD iscovalently attached to said surface usingN-sulphosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate.
 22. Themethod of claim 1, wherein said covalently attaching one or moreproteins or peptides in step b) further comprises: i) introducing saidcrosslinker or a solution containing said crosslinker to contact saidsurface and permitting said crosslinker or said solution containing saidcrosslinker to react with said surface; and ii) introducing at least oneprotein or peptide, or a solution containing at least one protein orpeptide to contact said surface.
 23. The method of claim 1, wherein saidcovalently attaching one or more proteins or peptides in b) furthercomprises exposing at least a portion of said surface to light.
 24. Themethod of claim 23, wherein said light comprises UV light.
 25. Themethod of claim 23, wherein said exposing comprises exposing a selectedarea or pattern for the covalent attachment of at least a portion ofsaid one or more proteins or peptides.
 26. The method of claim 23,wherein said exposing comprises masking said light so as to select saidselected area or pattern.
 27. The method of claim 23, wherein saidexposing comprises projecting a light pattern so as to select saidselected area or pattern.
 28. The method of claim 23, wherein saidexposing comprises rastering light so as to select said selected area orpattern.
 29. The method of claim 28, wherein said selected area orpattern comprises a linear pattern.
 30. The method of claim 28, whereinsaid cells comprise muscle cells or muscle-like cells that align withrespect to said selected area of pattern.
 31. The method of claim 1,wherein said covalently attaching one or more proteins or peptides in b)further comprises introducing said crosslinker to contact only one ormore selected areas of said microfluidic device.
 32. The method of claim1, wherein said microfluidic device further comprises a porous membrane.33. The method of claim 32, wherein said porous membrane comprises saidsurface.